All imagers, under ideal conditions and dimmed lighting were able to detect down to a surface concentration of for both IRDye 800CW and IRDye 680RD (for dual-channel systems that have this channel). Thus, per this criterion, all systems seem suitable for imaging high concentrations of ICG, as intended. For imaging lower concentrations of fluorophores, it is observed that systems with higher bit depths, variable electronic gain settings, and/or background-light correction during acquisition had the best sensitivity. The VisionSense Iridium system outperforms all other instruments in terms of sensitivity to low concentrations owing to its high camera bit depth and gain adjustment (1 to ) capability. The Solaris was a close second in terms of sensitivity, again due to the high bit depth and background correction functionality based on pulsing excitation light; but the lack of gain adjustment likely limits its sensitivity below . The Fluobeam800 system with the ability to manually vary exposure time could achieve sensitivity down to , but this comes at the expense of long-exposure times on the order of seconds, which may not be feasible within a clinical setting. Similar sensitivity is obtained with the Novadaq SPY system in real-time video mode. The Quest Spectrum though equipped with a 14-bit camera compresses the image data to 8-bits at the camera output resulting in reduced sensitivity and dynamic range, which severely affected the overall system performance. In terms of quantitative ability, the closer a log–log fitted-slope (Fit equation: which is equivalent to , where is the slope) was to 1, the more reliable a system could be for linear reporting of concentration, which was seen well with the Solaris system, owing to background correction. The Quest spectrum and VisionSense Iridium devices utilize 14-bit and 12-bit cameras, respectively, but ultimately map their data to 8-bit thus resulting in nonlinear compression and an observed slope of . While VisionSense uses smart image processing algorithms to produce a wide dynamic range, the Quest Spectrum lacked such features, thus the range of detection suffers, and sensitivity is about . It should be noted that for data from VisionSense and Fluobeam fluorescence signal measurements were scaled by the gain settings and exposure times, respectively. Aside from fluorescence intensity, some systems, e.g., Novadaq SPY, may also provide perfusion and flow rate as quantitative endpoints; these measurements are usually made in software based on the fluorescence intensity data, and would hence be affected by the linearity and quantitative performance of the system as described herein. Curadel’s Lab-Flare R1 was excluded from Fig. 4 as a final commercial system was unavailable for testing at the time of publishing this paper, although based upon the design the sensitivity, it was expected to be comparable to the other advanced systems and reported to be in the single nM range or better. The SurgVision system was also excluded from Fig. 4 as the sensitivity tests on this system were not available in the same methodology as used for all the other systems. However, the sensitivity to IRDye 800CW reported by the company is , which would make it among the most sensitive of the systems evaluated. This is most likely attributable to their use of an EMCCD capable of single-photon detection.